In the horse, it is important to make a reliable ECG recording during exercise as cardiac arrhythmia is one of the possible causes of poor performance. The purpose of the present study was to obtain a satisfactory, standardized method for ECG recording by computer during a standardized treadmill exercise test and to evaluate the procedure both in healthy and in diseased horses. Ten experienced healthy research horses were used to develop and adapt the original method (for humans) of computerized ECG recording with the Cardio Perfect software program and to obtain reference values for heart rate and blood variables. Further, three diseased horses (patients) were examined to evaluate whether the test procedures were also usable for horses without extensive experience in treadmill and other experimental procedures. During a standardized exercise test on a treadmill at walk (1.7 m/s), trot (4.0 m/s), and canter (7.0 m/s at 0% and 4% treadmill inclination), the ECG was recorded using two bipolar leads (from xiphoid to manubrium respectively to the left withers) and one unipolar lead (on the left ventral thorax). The quality of the exercise ECGs was satisfactory. The heart rate was detectable and arrhythmias were identifiable. Most research horses did not show arrhythmias during exercise, except a single atrial premature complex (APC). The reference values for the mean heart rate for the different stages of the exercise test differed from 84 + or - 7 to 176 + or - 21 beats per minute (bpm). The exercise test provoked in healthy horses a plasma lactate concentration around the anaerobic threshold. Most patients were also able to finish the test. In conclusion, the developed method to obtain an exercise ECG during a standardized exercise test proved to be reliable and applicable both in research horses and in patients, thus extending the possibilities to diagnose arrhythmias.